Biomedical subjects

S M Schuster

Publications and source records attributed to S M Schuster.

At least 19 recordsLinked to original sources

PubMed2001

Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B.

Asparagine synthetase catalyzes the ATP-dependent formation of L-asparagine from L-aspartate and L-glutamine, via a beta-aspartyl-AMP intermediate. Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme. Prior work showed that L-cysteine sulfinic acid competitively inhibits this enzyme by interfering with L-aspartate binding. Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E. coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of beta-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle. While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor. A sulfoximine reported recently by Koizumi et al. as a competitive inhibitor of the ammonia-dependent E. coli asparagine synthetase A (AS-A) [Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J. (1999) J. Am. Chem. Soc. 121, 5799-5800] can be regarded as such a species. We found that this sulfoximine also inhibited AS-B, effectively irreversibly. Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine. Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B.

Adenosine Monophosphate↗

PubMed2000

Activation of the human asparagine synthetase gene by the amino acid response and the endoplasmic reticulum stress response pathways occurs by common genomic elements.

The human asparagine synthetase (AS) gene is transcriptionally regulated by amino acid deprivation (amino acid response, AAR) and the endoplasmic reticulum stress response (ERSR), also known as the unfolded protein response pathway. The results reported here document the novel observation that induction of the AS gene by the AAR and ERSR pathways occurs via the same set of genomic elements. Data supporting this conclusion include transient transfection of AS promoter/reporter gene constructs that illustrate that the transcriptional control elements used by both pathways are contained with nucleotides -111 to -34 of the AS promoter. In vivo footprinting analysis of this region identified six specific protein-binding sites. Within two of these sites, altered footprinting was observed following amino acid or glucose deprivation, but the patterns were identical for both the AAR and the ERSR pathway. Site-directed mutation of individual nucleotides within these two binding sites confirmed their importance for regulated transcription, and none of the mutations resulted in loss of response of only one pathway. Neither of these two sites corresponds to a recently identified ERSR cis-element, nor do they contain consensus sequences for known transcription factors. Collectively, the data document that there are at least two independent transcriptional mechanisms for gene activation by the ERSR pathway, one of which terminates at the same genomic elements used by the AAR pathway.

Amino Acids↗

PubMed1999

Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product.

Asparagine synthetase B catalyzes the assembly of asparagine from aspartate, Mg(2+)ATP, and glutamine. Here, we describe the three-dimensional structure of the enzyme from Escherichia colidetermined and refined to 2.0 A resolution. Protein employed for this study was that of a site-directed mutant protein, Cys1Ala. Large crystals were grown in the presence of both glutamine and AMP. Each subunit of the dimeric protein folds into two distinct domains. The N-terminal region contains two layers of antiparallel beta-sheet with each layer containing six strands. Wedged between these layers of sheet is the active site responsible for the hydrolysis of glutamine. Key side chains employed for positioning the glutamine substrate within the binding pocket include Arg 49, Asn 74, Glu 76, and Asp 98. The C-terminal domain, responsible for the binding of both Mg(2+)ATP and aspartate, is dominated by a five-stranded parallel beta-sheet flanked on either side by alpha-helices. The AMP moiety is anchored to the protein via hydrogen bonds with O(gamma) of Ser 346 and the backbone carbonyl and amide groups of Val 272, Leu 232, and Gly 347. As observed for other amidotransferases, the two active sites are connected by a tunnel lined primarily with backbone atoms and hydrophobic and nonpolar amino acid residues. Strikingly, the three-dimensional architecture of the N-terminal domain of asparagine synthetase B is similar to that observed for glutamine phosphoribosylpyrophosphate amidotransferase while the molecular motif of the C-domain is reminiscent to that observed for GMP synthetase.

Adenosine Monophosphate↗

PubMed1999

Unusual amino acid usage in the variable regions of mercury-binding antibodies.

Monoclonal antibodies (mAb) specific for mercuric ions were isolated from BALB/c mice injected with a mercury-containing, hapten-carrier complex. The antibodies reacted by enzyme-linked immunosorbent assay with bovine serum albumin-glutathione-mercuric chloride (BSA-GSH-HgCl) but not with BSA-GSH without mercury. Nucleotide sequences from polymerase chain reaction products encoding six of the antibody heavy-chain variable regions and seven light-chain variable regions revealed that all the antibodies contained an unpaired cysteine residue in one hypervariable region, which is unusual for murine antibodies. Mutagenesis of the cysteine to either tyrosine or serine in one of the Hg-binding antibodies, mAb 4A10, eliminated mercury binding. However, of two influenza-specific antibodies that contain cysteine residues at the same position as mAb 4A10, one reacted with mercury, although not so strongly as 4A10, whereas the other did not react at all. These results suggested that, in addition to an unpaired cysteine, there are other structural features, not yet identified, that are important for creating an appropriate environment for mercury binding. The antibodies described here could be useful for investigating mechanisms of metal-protein interactions and for characterizing antibody responses to structurally simple haptens.

Amino Acid Sequence↗

PubMed1999

Activation of the unfolded protein response pathway induces human asparagine synthetase gene expression.

The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both amino acid and glucose deprivation of cells. The data reported here document that the human AS gene is induced following activation of the Unfolded Response Pathway (UPR), also known as the Endoplasmic Reticulum Stress Response (ERSR) in mammals. Increased AS transcription occurs in response to glucose deprivation, tunicamycin, or azetidine-2-carboxylate, all known to activate the UPR/ERSR pathway. Previously identified ERSR target genes contain multiple copies of a single highly conserved cis-element. In contrast, the human AS gene does not contain the ERSR element, as it has been described for other responsive genes. Instead, AS induction requires an Sp1-like sequence, a sequence previously shown to be associated with amino acid control of transcription, and possibly, a third region containing no consensus sequences for known transcription factors. Oligonucleotides covering each of these regions form DNA-protein complexes in vitro, and for some the amount of these complexes is greater when nuclear extracts from glucose-starved cells are tested. These results document that a wider range of metabolic activities are activated by the UPR/ERSR pathway than previously recognized and that genomic elements other than those already described can serve to enhance transcription of specific target genes.

Aspartate-Ammonia Ligase↗

PubMed1999

Transcriptional regulation of the human asparagine synthetase gene by carbohydrate availability.

Transcription of the asparagine synthetase (AS) gene is induced by amino acid deprivation. The present data illustrate that this gene is also under transcriptional control by carbohydrate availability. Incubation of human HepG2 hepatoma cells in glucose-free medium resulted in an increased AS mRNA content, reaching a maximum of about 14-fold over control cells after approx. 12 h. Extracellular glucose caused the repression of the content of AS mRNA in a concentration-dependent manner, with a k1/2 (concentration causing a half-maximal repression) of 1 mM. Fructose, galactose, mannose, 2-deoxyglucose and xylitol were found to maintain the mRNA content of both AS and the glucose-regulated protein GRP78 in a state of repression, whereas 3-O-methylglucose did not. Incubation in either histidine-free or glucose-free medium also resulted in adaptive regulation of the AS gene in BNL-CL.2 mouse hepatocytes, rat C6 glioma cells and human MOLT4 lymphocytes, in addition to HepG2 cells. In contrast, the steady-state mRNA content of GRP78 was unaffected by amino acid availability. Transient transfection assays using a reporter gene construct documented that glucose deprivation increases AS gene transcription via elements within the proximal 3 kbp of the AS promoter. These results illustrate that human AS gene transcription is induced following glucose limitation of the cells.

Animals↗

PubMed1999

Formation and isolation of a covalent intermediate during the glutaminase reaction of a class II amidotransferase.

Incubation of Escherichia coli asparagine synthetase B (AS-B) with [14C]-L-glutamine gives a covalent adduct that can be isolated. Radiolabeled protein is not observed (i) when the wild-type enzyme is incubated with 6-diazo-5-oxo-L-norleucine (DON) prior to reaction with [14C]glutamine or (ii) when the C1A AS-B mutant is incubated with [14C]-L-glutamine. Both of these alterations eliminate the ability of the enzyme to utilize glutamine but do not affect ammonia-dependent asparagine synthesis. Formation of the covalent adduct therefore depends on the presence of the N-terminal active site cysteine, which has been shown to be essential for glutamine-dependent activity in this and other class II amidotransferases. The amount of covalent adduct exhibits saturation behavior with increasing concentrations of L-glutamine. The maximum observed quantity of this intermediate is consistent with its involvement on the main pathway of glutamine hydrolysis. The chemical properties of the isolable covalent adduct are consistent with those anticipated for the gamma-glutamyl thioester that has been proposed as an intermediate in the AS-B-catalyzed conversion of glutamine to glutamate. The covalent adduct is acid-stable but is labile under alkaline conditions. On the basis of the measured rates of formation and breakdown of this intermediate, it is kinetically competent to participate in the normal catalytic mechanism. These studies represent the first description of a thioester intermediate for any class II amidotransferase and represent an important step in gaining further insight into the kinetic and chemical mechanisms of AS-B.

Aspartate-Ammonia Ligase↗

PubMed1998

Kinetic mechanism of Escherichia coli asparagine synthetase B.

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartate, glutamine, and ATP. A combination of kinetic, isotopic-labeling, and stoichiometry studies have been performed to define the nature of nitrogen transfer mediated by AS-B. The results of initial rate studies were consistent with initial binding and hydrolysis of glutamine to glutamate plus enzyme-bound ammonia. The initial velocity results were equally consistent with initial binding of ATP and aspartate prior to glutamine binding. However, product inhibition studies were only consistent with the latter pathway. Moreover, isotope-trapping studies confirmed that the enzyme-ATP-aspartate complex was kinetically competent. Studies using 18O-labeled aspartate were consistent with formation of a beta-aspartyl-AMP intermediate, and stoichiometry studies revealed that 1 equiv of this intermediate formed on the enzyme in the absence of a nitrogen source. Taken together, our results are most consistent with initial formation of beta -aspartyl-AMP intermediate prior to glutamine binding. This sequence leaves open many possibilities for the chemical mechanism of nitrogen transfer.

Adenosine Monophosphate↗

PubMed1998

Mechanistic issues in asparagine synthetase catalysis.

The enzymatic synthesis of asparagine is an ATP-dependent process that utilizes the nitrogen atom derived from either glutamine or ammonia. Despite a long history of kinetic and mechanistic investigation, there is no universally accepted catalytic mechanism for this seemingly straightforward carboxyl group activating enzyme, especially as regards those steps immediately preceding amide bond formation. This chapter considers four issues dealing with the mechanism: (a) the structural organization of the active site(s) partaking in glutamine utilization and aspartate activation; (b) the relationship of asparagine synthetase to other amidotransferases; (c) the way in which ATP is used to activate the beta-carboxyl group; and (d) the detailed mechanism by which nitrogen is transferred.

Adenosine Triphosphate↗

PubMed1998

Antimalarial activities of polyhydroxyphenyl and hydroxamic acid derivatives.

Several known mammalian ribonucleotide reductase inhibitors featuring a polyhydroxyphenyl and/or hydroxamate moiety as the active group were screened for potency in inhibiting growth of the malaria parasite Plasmodium falciparum. Compounds containing a 2,3- or 3,4-dihydroxyphenyl group as well as benzohydroxamate appear to be the most effective inhibitors of the malaria parasite.

Animals↗

PubMed1997

Identification of cysteine-523 in the aspartate binding site of Escherichia coli asparagine synthetase B.

The site-directed chemical modifier [p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA) inactivates Escherichia coli asparagine synthetase B activity following pseudo-first-order kinetics, with ATP providing specific protection, with a Kd of 12 microM. The 5'-FSBA modification appears to be covalent, even though a nonstoichiometric amount (less than 10%) of radiolabeled 5'-FSBA was associated with a totally inactivated enzyme. However, the inactivation by 5'-FSBA could be reversed upon the addition of dithiothreitol. These results are indicative of 5'-FSBA-induced disulfide bond formation, which requires the presence of at least two cysteine residues in the proximity of the ATP binding site. Identification of the critical cysteine residue was accomplished by sequential replacement of each cysteine in the protein by site-directed mutagenesis. Cys 523 was identified as the key residue involved in the formation of the 5'-FSBA-induced disulfide bond. Detailed kinetic analyses and comparison with similar enzymes, suggest that this cysteine residue, while in close proximity to the ATP binding site, is actually involved in aspartate binding in asparagine synthetase B.

Adenosine↗

PubMed1997

Mutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by Escherichia coli asparagine synthetase B.

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.

Adenosine Monophosphate↗

PubMed1997

Variations of hepatitis B virus core gene sequence in Western patients with chronic hepatitis B virus infection.

Heterogeneity of the hepatitis B virus (HBV) core gene has been reported to be associated with the presence of active liver disease in Japanese patients with chronic HBV infection. This study evaluated the significance of HBV core gene heterogeneity in Western patients with chronic HBV infection. The hepatitis B virus precore/core gene from 45 patients (inactive:active liver disease ratio 16:29) was amplified from serum by polymerase chain reaction (PCR). Gel electrophoresis was employed to detect large deletions. The PCR amplicons from 13 patients (all HBV serotype adw but with a different spectrum of liver disease) were cloned and sequenced. Hepatitis B surface antigen (HBsAg) serotypes were tested by enzyme immunoassay (EIA) and hepatic expression of HBV antigens was assessed by immunohistochemistry. The HBV core gene was amplified from the serum of all 45 patients. Three patients had mixed infection with both precore mutant and wild-type HBV and all three had active liver disease. No patient had a large deletion of the HBV core gene. Hepatitis B virus core gene sequence variations were more common in the midcore region and there was no difference in the number of silent and missense substitutions between those with inactive and active liver disease. There was no correlation between the nucleotide or encoded amino acid substitutions and the clinical and biochemical parameters, including the subsequent response to interferon-alpha therapy (n = 37) or hepatic HBV antigen expression. Variation of the HBV core gene was not found to be preferentially associated with active liver disease in Western patients with chronic HBV infection. The pattern of hepatitis B core gene variation is in accord with the genomic organization of HBV.

Adult↗

PubMed1997

Amino acid control of asparagine synthetase: relation to asparaginase resistance in human leukemia cells.

Complete amino acid deprivation in mammalian cells causes a significant enhancement in gene expression for a number of important cellular activities; among these is asparagine synthetase (AS). The data presented demonstrate that, in both nonleukemic (rat Fao hepatoma cells) and human leukemia cells (MOLT-4, NALL-1, and BALL-1), AS mRNA levels, protein content, and enzymatic activity are induced after incubation in an otherwise complete tissue culture medium that is deficient in a single amino acid or in medium that has been depleted of the amino acid asparagine by the addition of asparaginase. Complete amino acid deprivation results in a concerted increase in AS mRNA, protein, and enzymatic activity, which, in conjunction with previously published research, suggests that the mechanism of this cellular response involves transcriptional control of the AS gene. Asparaginase treatment is a standard component of acute lymphoblastic leukemia therapy for which the effectiveness is related to the inability of these cells to upregulate AS activity to a sufficient level. With regard to the asparaginase sensitivity of the three human leukemia cell lines, there was a trend toward an inverse relation to the degree of AS expression. Selection for asparaginase-resistant MOLT-4 sublines resulted in enhanced AS mRNA and protein content regardless of whether the cells had been selected by asparaginase treatment directly or asparagine was removed from the culture medium. Collectively, the data illustrate that further advances in asparaginase therapy will require additional knowledge of amino acid-dependent regulation of AS gene expression and, conversely, that asparaginase resistance represents a model system for investigating metabolite control in a clinically relevant setting.

Amino Acids↗

PubMed1996

Mapping the aspartic acid binding site of Escherichia coli asparagine synthetase B using substrate analogs.

Novel inhibitors of asparagine synthetase, that will lower circulating levels of blood asparagine, have considerable potential in developing new protocols for the treatment of acute lymphoblastic leukemia. We now report the indirect characterization of the aspartate binding site of Escherichia coli asparagine synthetase B (AS-B) using a number of stereochemically, and conformationally, defined aspartic acid analogs. Two compounds, prepared using novel reaction conditions for the stereospecific beta-functionalization of aspartic acid diesters, have been found to be competitive inhibitors with respect to aspartate in kinetic studies on AS-B. Chemical modification experiments employing [(fluorosulfonyl)benzoyl]adenosine (FSBA), an ATP analog, demonstrate that both inhibitors bind to the aspartate binding site of AS-B. Our results reveal that large steric alterations in the substrate are not tolerated by the enzyme, consistent with the failure of previous efforts to develop AS inhibitors using random screening approaches, and that all of the ionizable groups are placed in close proximity in the bound conformation of aspartate.

Alkylation↗

PubMed1996

Probing the mechanism of nitrogen transfer in Escherichia coli asparagine synthetase by using heavy atom isotope effects.

In experiments aimed at determining the mechanism of nitrogen transfer in purF amidotransferase enzymes, 13C and 15N kinetic isotope effects have been measured for both of the glutamine-dependent activities of Escherichia coli asparagine synthetase B (AS-B). For the glutaminase reaction catalyzed by AS-B at pH 8.0, substitution heavy atom labels in the side chain amide of the substrate yields observed values of 1.0245 and 1.0095 for the amide carbon and amide nitrogen isotope effects, respectively. In the glutamine-dependent synthesis of asparagine at pH 8.0, the amide carbon and amide nitrogen isotope effects have values of 1.0231 and 1.0222, respectively. We interpret these results to mean that nitrogen transfer does not proceed by the formation of free ammonia in the active site of the enzyme and probably involves a series of intermediates in which glutamine becomes covalently attached to aspartate. While a number of mechanisms are consistent with the observed isotope effects, a likely reaction pathway involves reaction of an oxyanion with beta-aspartyl-AMP. This yields an intermediate in which C-N bond cleavage gives an acylthioenzyme and a second tetrahedral intermediate. Loss of AMP from the latter gives asparagine. An alternate reaction mechanism in which asparagine is generated from an imide intermediate also appears consistent with the observed kinetic isotope effects.

Aspartate-Ammonia Ligase↗

PubMed1996

Glutamic acid gamma-monohydroxamate and hydroxylamine are alternate substrates for Escherichia coli asparagine synthetase B.

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartic acid and glutamine in an ATP-dependent reaction. The ability of this enzyme to employ hydroxylamine and L-glutamic acid gamma-monohydroxamate (LGH) as alternative substrates in place of ammonia and L-glutamine, respectively, has been investigated. The enzyme is able to function as an amidohydrolase, liberating hydroxylamine from LGH with high catalytic efficiency, as measured by k(cat)/K(M). In addition, the kinetic parameters determined for hydroxylamine in AS-B synthetase activity are very similar to those of ammonia. Nitrogen transfer from LGH to yield aspartic acid beta-monohydroxamate is also catalyzed by AS-B. While such an observation has been made for a few members of the trpG amidotransferase family, our results appear to be the first demonstration that nitrogen transfer can occur from glutamine analogs in a purF amidotransferase. However, k(cat)/K(M) for the ATP-dependent transfer of hydroxylamine from LGH to aspartic acid is reduced 3-fold relative to that for glutamine-dependent asparagine synthesis. Further, the AS-B mutant in which asparagine is replaced by alanine (N74A) can also use hydroxylamine as an alternate substrate to ammonia and catalyze the hydrolysis of LGH. The catalytic efficiencies (k(cat)/K(M)) of nitrogen transfer from LGH and L-glutamine to beta-aspartyl-AMP are almost identical for the N74A AS-B mutant. These observations support the proposal that Asn-74 plays a role in catalyzing glutamine-dependent nitrogen transfer. We interpret our kinetic data as further evidence against ammonia-mediated nitrogen transfer from glutamine in the purF amidotransferase AS-B. These results are consistent with two alternate chemical mechanisms that have been proposed for this reaction [Boehlein, S. K., Richards, N. G. J., Walworth, E. S., & Schuster, S. M. (1994) J. Biol. Chem. 269, 26789-26795].

Aspartate Aminotransferases↗

PubMed1996

Histidine-49 is necessary for the pH-dependent transition between active and inactive states of the bovine F1-ATPase inhibitor protein.

The role of the histidyl residue at position 49 (H49) of the bovine mitochondrial F1-ATPase inhibitor protein (F1I) was examined by site-directed mutagenesis. Six amino acids (Q, E, K, V, L, and I) were substituted for H49 and the activities of the resulting inhibitor proteins were characterized with respect to pH. Each of the six mutations abolished the pH sensitivity which is characteristic of wild-type F1I. At pH 8.0 each of the mutations caused an increase in apparent maximum inhibition and a decrease in apparent Ki relative to wild type. At pH 6.7 the hydrophilic substitutions had little effect on apparent Ki, while the hydrophobic substitutions caused increases of 3.5- to 8.5-fold relative to wild type. The ratios of apparent Ki at pH 8.0 to apparent Ki at pH 6.7 were in the range of 0.5 to 1.6 for the mutants, whereas the wild-type value is 15.0. The mutations appear to shift the equilibrium between active and inactive conformations of F1I toward the active state. We find that H49 is required by F1I for sensitivity to pH and that it may facilitate the transition between active and inactive states of F1I. A possible role for H49 in the stabilization of the inactive state through participation in a multivalent complex with Zn2+ is also discussed.

Amino Acid Sequence↗